We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.